Listeria monocytogenes is a facultative intra-cytoplasmic bacterial pathogen that is an important agent of serious human food-borne infections. As a bacterium commonly found in the environment, L. monocytogenes is able to mediate the complex transition from life in the soil to life in human cells where it survives and replicates. This proposal seeks to determine what is required for an environmental bacterium to mediate the switch from outside life in the soil to life inside of mammalian cells. On-going work has focused on a key transcriptional activator of virulence gene expression in L. monocytogenes known as PrfA. PrfA is required for the expression of nearly every L monocytogenes virulence factor identified to date, however the mechanisms used by L. monocytogenes to coordinate the expression of gene products within host cells are unknown. PrfA exists in high activity and low activity states, and the working hypothesis of this proposal is that the regulation of PrfA activation is critical for L. monocytogenes to successfully transition from the outside environment to life within mammalian cells. This proposal will use a combination of genetic and biochemical approaches to elucidate the mechanisms by which L. monocytogenes regulates the expression of virulence genes within host cells. It will examine the pathogenic consequences of PrfA activation, and will define bacterial gene products that are induced within host cells and that are directly or indirectly dependent upon PrfA for expression. Aim 1 will focus on the biochemical analysis of mutationally activated forms of PrfA and assessment of the impact of PrfA activation on L. monocytogenes physiology and pathogenesis. Aim 2 will elucidate the contributions of newly identified PrfA-dependent gene products to L. monocytogenes pathogenesis. Aim 3 undertakes functional characterization of mutations located outside of the prfA locus that alter the regulation of L monocytogenes virulence gene expression. The ultimate goal of the proposed work is to elucidate the regulatory pathways that enable bacterial survival within host cells, and the functional identification of additional bacterial factors that support replication within the host.